Dinh/Dinh 2014/NOTES/2014-3-11: Difference between revisions
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The best annealing temperature appears to be 56.4 degC. This temperature should apply to all the subsets since the probes were randomly assigned to subsets. | The best annealing temperature appears to be 56.4 degC. This temperature should apply to all the subsets since the probes were randomly assigned to subsets. | ||
* Will try capture subset B, C, D, E, and F at 56.4 degC. Also, I will amplify 19 or 20 cycles to hopefully increase yield of capture amplicons. | * Will try capture subset B, C, D, E, and F at 56.4 degC. Also, I will amplify 19 or 20 cycles to hopefully increase yield of capture amplicons. | ||
* TBE Gel | * TBE Gel for capture at 56.4 (sample conditions), I added 10 uL water during ExoI/ExoIII and amplified 4 uL for 20 cycles. | ||
[[File:HOTSPOTS_captureA_E_newTemp.jpg | 300px]] | [[File:HOTSPOTS_captureA_E_newTemp.jpg | 300px]] |
Revision as of 05:45, 27 March 2014
HOTSPOTS 460K Capture
Capture calculations
Size of Probes | 90000 | units |
gDNA | 150 | ng |
Human (MW) | 1.82E+012 | g/moles |
1 Probe (MW) | 31360.1 | g/moles |
Template | 8.23E-011 | nmoles |
Probes(100:1) | 8.23E-009 | nmoles |
Amount Probes needed | 23.23 | ng |
Size of Probes | 6553 | units |
gDNA | 150 | ng |
Human (MW) | 1.82E+012 | g/moles |
1 Probe (MW) | 31360.1 | g/moles |
Template | 8.23E-011 | nmoles |
Probes(100:1) | 8.23E-009 | nmoles |
Amount Probes needed | 1.69 | ng |
Human MW = (3x109 bp)*(607.4 gmoles-1/bp) + 157.9 gmoles-1 1 probe MW = (103 bp)*(303.7 gmoles-1)+ 79 gmoles-1 Amount probes = (1 probe MW) * (Size of probes) * (Moles of probes required)
Prepared probes
Subset | Dilution | QUBIT(ng/mL) | Conc(ng/uL) |
A | 100 | 81.2 | 8.12 |
B | 100 | 46.4 | 4.64 |
C | 100 | 84.8 | 8.48 |
D | 100 | 113 | 11.3 |
E | 100 | 84.7 | 8.47 |
F | 100 | 97.4 | 9.74 |
Test capture - 4 reactions
Pool subset probes for 4 reactions
Subset | Dilution | QUBIT(ng/mL) | Conc(ng/uL) | Volume(uL) | Amount for 4.5 capture (ng) | Subset Size |
A | 100 | 81.2 | 8.12 | 12.88 | 104.55 | 90000 |
B | 100 | 46.4 | 4.64 | 22.53 | 104.55 | 90000 |
C | 100 | 84.8 | 8.48 | 12.33 | 104.55 | 90000 |
D | 100 | 113 | 11.3 | 9.25 | 104.55 | 90000 |
E | 100 | 84.7 | 8.47 | 12.34 | 104.55 | 90000 |
F | 100 | 97.4 | 9.74 | 0.78 | 7.61 | 6553 |
- Pool probes for 4.5 captures and aliquot 15.58 uL per capture.
Capture setup
- I used up all the bisulfite converted gDNA because the converted DNA cannot be kept for long period of times.
Tube ID | Converted sample | Amount sample (ng) | Volume sample (uL) | Volume probes (uL) | Volume of 10x ampligase buffer | Volume water (uL) | Total volume |
1. 54 Ctrl | H1 p54 Control TeSR | 193.90 | 7.00 | 16.00 | 3.00 | 4.00 | 30 |
2. Act | H1 p54 Activin-A | 362.00 | 10.00 | 16.00 | 3.00 | 1.00 | 30 |
3. 47 Ctrl | H1 p47 Control TeSR | 144.95 | 6.50 | 16.00 | 3.00 | 4.50 | 30 |
4. Bmp | H1 p47 BMP-4 | 205.60 | 8.00 | 16.00 | 3.00 | 3.00 | 30 |
Capture program
1. 95°C 30s 2. 94.8°C 30s -0.2°C per 30s 198 times 3. 55°C 20h ADD 2.5 uL SLN mix 4. 55°C 19.5h 5. 94°C 2m ADD 2.0 uL ExoI/ExoIII mix 6. 37°C 2h 7. 90°C 5m 8. 4°C forever
Capture gel
- Amplified 4 uL in 25 uL reaction using KAPA 2xMM with AmpR6.3Ind17
REAGENT 1x 5.5x Capture template 4.00 uL -- 10 uM AmpF6.4Sol 0.50 uL 2.75 uL 10 uM AmpF6.3RInd17 0.50 uL 2.75 uL 2xKAPA Master Mix 12.50 uL 68.75 uL Nuclease free H2O 7.50 uL 41.25 uL
- TBE Gel:
File:HOTSPOTS capture1 test.jpg
Test capture subset A
Capture setup
Tube ID | Converted sample | Amount sample (ng) | Volume sample (uL) | Volume probes (uL) | Volume of 10x ampligase buffer | Volume water (uL) | Total volume |
GM-A-1-55 | GM12878 | 150.0 | 2.400 | 6.00 | 2.00 | 9.60 | 20.00 |
GM-A-5-56.4 | GM12878 | 150.0 | 2.400 | 6.00 | 2.00 | 9.60 | 20.00 |
GM-A-8-58.8 | GM12878 | 150.0 | 2.400 | 6.00 | 2.00 | 9.60 | 20.00 |
GM-A-12-60 | GM12878 | 150.0 | 2.400 | 6.00 | 2.00 | 9.60 | 20.00 |
Capture program
1. 95°C 30s 2. 94.8°C 30s -0.2°C per 30s 174 times -> 60°C 3. Gradient: 55/56.4/58.8/60 °C 20h ADD 2.5 uL SLN mix 4. Gradient: 55/56.4/58.8/60 °C 20h 5. 94°C 2m ADD 2.0 uL ExoI/ExoIII mix 6. 37°C 2h 7. 90°C 5m 8. 4°C forever
Capture PCR
- 4 uL of capture was used to amplify. Two step PCR with 8 cycles first step, and 10 cycles second step = 18 cycles of PCR total for all samples.
- TBE Gel:
File:HOTSPOTS captureA test.jpg File:HOTSPOTS captureA test repeat.job Gel might be slightly shifted or 471 bp calculation is wrong? The expected band seems to be greater than 500bp. The best annealing temperature appears to be 56.4 degC. This temperature should apply to all the subsets since the probes were randomly assigned to subsets.
- Will try capture subset B, C, D, E, and F at 56.4 degC. Also, I will amplify 19 or 20 cycles to hopefully increase yield of capture amplicons.
- TBE Gel for capture at 56.4 (sample conditions), I added 10 uL water during ExoI/ExoIII and amplified 4 uL for 20 cycles.
File:HOTSPOTS captureA E newTemp.jpg